绕道聚合酶的动力学和热稳定性,DinB同类物 (Dbh)
Jenaro Soto1, Sean L Moro2, Melanie J Cocco1,2
1Department of Pharmaceutical Sciences, University of California, Irvine, CA, United States.
Frontiers in molecular biosciences
|May 15, 2024
概括
DinB同类聚合酶 (Dbh) 在更高的温度下显示出增强的动态,与晶体结构不同. 这种Y家族DNA聚合酶在溶液中表现出冷变性和独特的金属离子结合点.
科学领域:
- 生物化学和分子生物学
- 结构生物学 结构生物学
- 考古生物学 考古生物学
背景情况:
- 丁B同类聚合酶 (Dbh) 是来自热爱古生物*Sulfolobus acidocaldarius*的Y家族转化DNA聚合酶.
- 对于Dbh的现有结构数据是在低活动温度下获得的,不反映其功能范围.
- 了解Dbh的温度依赖的结构和动态对于其在DNA修复中的功能至关重要.
研究的目的:
- 研究温度 (2°C-65°C) 和金属离子结合对Dbh.的溶液结构和动态的影响.
- 将溶液状态结构和动态特性与现有的固态 (X射线) 结构进行比较.
- 在没有DNA的情况下,在Dbh中识别金属离子结合点.
主要方法:
- 核磁共振 (NMR) 光谱,包括交换保护因子和温度系数.
- 循环二元化 (CD) 光谱法用于评估三级结构变化.
- 化学转移扰动分析用于绘制金属离子结合点的地图.
主要成果:
- 随着温度的上升,Dbh表现出增加的域动态 (指,手指,LF),特别是在35°C和50°C之间.
- 核磁共振显示Dbh的三级结构从25°C开始冷变质,在较低的温度下变质增加.
- 使用Mg2+和Mn2+在溶液中确定了三个金属离子结合点,其中两个靠近活性点,一个在LF域中,这些结合点在晶体结构中没有观察到.
结论:
- Dbh的溶液结构和动态与其报告的晶体结构不同,特别是在温度效应和金属离子协调方面.
- Dbh表现出显著的冷变性,表明热友酶具有独特的结构特征.
- 已确定的金属结合点,特别是LF领域的新型结合点,可能在Dbh的催化活动或调节中发挥作用.
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